# How to open .czi, .nd2, .lif and other microscope files in ImageJ (Bio-Formats)

> Open Zeiss .czi, Nikon .nd2 and Leica .lif files in ImageJ with Bio-Formats (built into Fiji), keep the pixel size, and fix "Unsupported format".

Published 2026-11-06 by LabCanvas team. https://www.labcanvas.com/blog/open-czi-nd2-files-imagej

![ImageJ 1.54f Image › Properties on a synthetic 16-bit nuclei image saved as TIFF: pixel width and height 0.325 micron read from the file, and the title bar giving 166.40 × 166.40 microns](https://www.labcanvas.com/blog/imagej/open-czi-nd2-files-imagej-properties.webp)

To open a .czi (Zeiss), .nd2 (Nikon), .lif (Leica) or other microscope file in ImageJ, use **Bio-Formats**: in Fiji it is built in, so drag the file onto the Fiji window or choose **File › Import › Bio-Formats**, set **View stack with: Hyperstack** and click **OK**. Channels, slices, time points and the pixel size come in from the file's metadata. Plain ImageJ 1.x needs Bio-Formats added first (one .jar in the plugins folder); without it, it stops with "Unsupported format or file not found".

To pass the image on with its scale intact, save it as **TIFF** or **OME-TIFF**, never as JPEG or PNG.

## What Bio-Formats is

Bio-Formats is an open-source library from the Open Microscopy Environment (OME) that reads well over a hundred life-science file formats and turns their metadata into one common model, OME (Linkert et al. 2010). In ImageJ it appears as a set of plugins: the **Bio-Formats Importer**, the **Bio-Formats Exporter**, a windowless importer that reuses your last settings, and a few helpers.

| File | Made by | Opens in Fiji | Opens in plain ImageJ 1.x |
|---|---|---|---|
| .czi | Zeiss ZEN | Yes (Bio-Formats) | Only after installing Bio-Formats |
| .nd2 | Nikon NIS-Elements | Yes (Bio-Formats) | Only after installing Bio-Formats |
| .lif, .lof | Leica LAS X | Yes (Bio-Formats) | Only after installing Bio-Formats |
| .oib, .oir, .vsi | Olympus / Evident | Yes (Bio-Formats) | Only after installing Bio-Formats |
| .lsm | Zeiss (older confocals) | Yes | Only after installing Bio-Formats |
| .ims | Bitplane Imaris | Yes (Bio-Formats) | Only after installing Bio-Formats |
| .tif, OME-TIFF | Any | Yes | Yes (OME metadata needs Bio-Formats) |

## How to open .czi, .nd2 and .lif files in Fiji

1. **Open the file.** Drag it onto the Fiji main window, or use **File › Open…**: Fiji hands files it cannot read itself to Bio-Formats. To see every option, use **File › Import › Bio-Formats** instead.
2. **Fill in the Bio-Formats Import Options** (the settings below). Fiji remembers them for next time.
3. **Choose the series**, if asked. A .lif project, a multi-position .nd2 or a .czi with several scenes holds many images; a second dialog lists them so you can tick the ones to open.
4. **Check the scale** with **Image › Properties…**: pixel width and height in µm, and the right numbers of channels (c), slices (z) and frames (t).

### The Bio-Formats Import Options that matter

| Option | What it does | Usual choice |
|---|---|---|
| View stack with | The viewer: **Hyperstack** (default), Standard ImageJ (old macros only), Data Browser, Image5D, View5D, or Metadata only | Hyperstack |
| Stack order | The order of the planes, XYCZT by default | Leave as is |
| Color mode | **Default** (as stored), **Composite** (channels merged), **Colorized**, **Grayscale**, **Custom** | Composite for a merge, Grayscale to measure |
| Autoscale | Sets the display range from the data | On; it changes only the display, not the pixel values |
| Split channels / Split focal planes / Split timepoints | Each channel, plane or time point as its own window | Off, unless you want separate images |
| Open all series | Opens every image in the file without asking | Off for a big .lif; pick series instead |
| Concatenate series when compatible | Joins series of the same size into one stack | For tiles or positions you want together |
| Use virtual stack | Reads planes from disk as you view them | On for files larger than your memory |
| Specify range for each series | Opens only some channels, slices or frames | For huge time-lapses |
| Crop on import | Opens only a region | For large tiled scans |
| Display metadata / Display OME-XML metadata | Shows the acquisition settings in a table | When you need the objective, exposure or laser lines |
| Group files with similar names | Opens a numbered series of files as one dataset | For one-file-per-plane exports |

The same importer can run from a macro; the Macro Recorder (**Plugins › Macros › Record…**) writes the exact line for your settings, for example `run("Bio-Formats Importer", "open=/path/to/file.czi autoscale color_mode=Default view=Hyperstack stack_order=XYCZT");`.

## How to install Bio-Formats in plain ImageJ 1.x

Fiji users can skip this: Bio-Formats is already there and the Fiji Updater (**Help › Update…**) keeps it current. For plain ImageJ 1.x:

1. Download **bioformats_package.jar** from the Bio-Formats download page on the OME website (openmicroscopy.org).
2. Put it in the **plugins** folder inside your ImageJ folder (on a Mac, ImageJ.app's plugins folder; **File › Show Folder › Plugins** opens it).
3. Restart ImageJ. A **Plugins › Bio-Formats** submenu appears, with **Bio-Formats Importer** and **Bio-Formats Exporter**, and **File › Open** now passes unknown formats to Bio-Formats.

The Bio-Formats documentation warns about one common mistake: two copies of bioformats_package.jar in the plugins folder. Delete the old one before you add a new one.

## "Unsupported format or file not found": what it means

Plain ImageJ 1.x reads TIFF, JPEG, PNG, GIF, BMP, DICOM, FITS, PGM, AVI and a few more on its own. Give it anything else without Bio-Formats and it stops with an error. The wording depends on how the file was opened; the two messages in ImageJ 1.54f are "Unsupported format or file not found" and "Format not supported or reader plugin not found".

![ImageJ error dialog: Unsupported format or file not found, for a file named Experiment_01.nd2](https://www.labcanvas.com/blog/imagej/open-czi-nd2-files-imagej-unsupported-format.webp "Plain ImageJ 1.54f with no Bio-Formats, opening a stand-in file with the .nd2 extension: Unsupported format or file not found 1. The folder part of the path is shortened here.")

Work through it in this order:

1. **Is the path right?** The same message appears when the file does not exist (a network drive that dropped, a file still syncing from the cloud).
2. **Is Bio-Formats installed?** In plain ImageJ, look for **Plugins › Bio-Formats**. If it is missing, install it as above, or use Fiji.
3. **Is it up to date?** New acquisition software versions sometimes write files an old Bio-Formats cannot read. In Fiji, run **Help › Update…**; in plain ImageJ, replace the jar.
4. **Still failing?** Export the image as OME-TIFF from the acquisition software and open that.

## How to keep the scale: check it, then save as TIFF or OME-TIFF

A microscope file carries its pixel size, and most measurements depend on it. Check it once the image is open.

**Image › Properties…** shows the pixel width and height with their unit, and the numbers of channels, slices and frames. **Image › Show Info…** lists the same and more: the size in µm, the resolution, the bit depth and the range of values.

![ImageJ Image Properties dialog showing a pixel width of 0.325 micron for a TIFF](https://www.labcanvas.com/blog/imagej/open-czi-nd2-files-imagej-properties.webp "Plain ImageJ 1.54f on the synthetic nuclei sample saved as a TIFF: Image › Properties shows 0.325 µm per pixel, read from the file 1, and the title bar gives the field size, 166.40 × 166.40 microns 2.")

![ImageJ Show Info window listing width and height in microns, resolution, pixel size and bit depth](https://www.labcanvas.com/blog/imagej/open-czi-nd2-files-imagej-show-info.webp "Image › Show Info on the same TIFF: 166.4 µm wide and high 1, 3.0769 pixels per micron, so 0.325 µm pixels 2, and still 16-bit 3.")

Now the same image saved as JPEG and opened again. The pixels look the same, but the scale and the bit depth are gone:

![ImageJ Image Properties dialog for the same image saved as JPEG: pixel width 1, unit pixel](https://www.labcanvas.com/blog/imagej/open-czi-nd2-files-imagej-properties-jpeg.webp "The same nuclei image after File › Save As › Jpeg and reopening it in ImageJ 1.54f: 1 pixel = 1 pixel 1, and 512 × 512 pixels, 8-bit in the title bar 2. The 16-bit values and the 0.325 µm scale are lost, and JPEG compression has changed the pixel values.")

So, to pass an image from a .czi or .nd2 to a colleague, a figure or another program:

- **File › Save As › Tiff…** in ImageJ or Fiji writes an ImageJ TIFF: raw values, the hyperstack layout, the pixel size, slice spacing and frame interval in the file's description. ImageJ and Fiji read it back unchanged.
- **OME-TIFF** is the open standard that other programs read too (OMERO, napari, QuPath, CellProfiler, Python). In Fiji, use **Plugins › Bio-Formats › Bio-Formats Exporter** and give the file a name ending in **.ome.tif**. Bio-Formats also has a command-line converter, **bfconvert**, for whole folders.
- **Not** JPEG or PNG for anything you will measure: they hold 8-bit pictures without a calibration.

## Pitfalls

- **The scale did not come through.** If **Image › Properties** shows 1 pixel = 1 pixel after import, the file had no calibration or the reader could not find it. Set it from the objective and camera (the [pixel size calculator](https://www.labcanvas.com/tools/microscope-pixel-size-calculator) does the sums) or from a stage micrometer; see [how to set the scale in ImageJ](https://www.labcanvas.com/blog/imagej-scale-bar-set-scale).
- **Slices and frames swapped.** A time-lapse that opens as a Z-stack makes every per-frame tool wrong. Fix it in **Image › Properties** by swapping the numbers of slices and frames, or with **Image › Hyperstacks › Stack to Hyperstack…** for a plain stack.
- **Autoscale is not data.** Autoscale and Color mode change the display only. Measure on the raw values, and never measure on a copy flattened to RGB.
- **The wrong series.** A .lif or a slide scan can hold dozens of series, including thumbnails, labels or overview images at lower resolution. Check the size and pixel size of the series you opened.
- **Running out of memory.** A big file can be larger than ImageJ's memory limit. Tick **Use virtual stack**, open a range of planes, or raise the limit in **Edit › Options › Memory & Threads…**.
- **A screenshot is not an image.** Copying from the vendor viewer or exporting "as displayed" gives an 8-bit RGB picture with contrast already applied. Export the raw data.

## Reporting it

A methods sentence to copy, with the parts in brackets filled in:

> Images were acquired as [.czi] files and opened in [Fiji (ImageJ 1.54f)] with the Bio-Formats importer [version], which read the pixel size ([0.325] µm) from the file metadata. Images were analysed on the raw [16-bit] data and exported as OME-TIFF for [figure preparation / analysis].

Cite Bio-Formats (Linkert et al. 2010) and Fiji (Schindelin et al. 2012). Keep the original vendor files: they hold the full acquisition metadata (objective, exposure, laser lines), and journals and repositories increasingly ask for them.

## An easier way: measure the exported TIFF in LabCanvas

Once the file is a TIFF or OME-TIFF, you can measure it in [LabCanvas Image Analysis](https://www.labcanvas.com/analysis.html), in the browser with nothing to install. It does not read vendor formats: .czi, .nd2, .lif, .lsm, .oib, .vsi and .ims need exporting first, as above.

1. Export from Fiji (**File › Save As › Tiff…**, or the Bio-Formats Exporter for .ome.tif) or from the acquisition software as OME-TIFF.
2. Open [Image Analysis](https://www.labcanvas.com/analysis.html) and drop the file on the window. Channels, slices, frames, pixel size, slice spacing, frame interval and channel names come in from the file.
3. Read the image check: it reports the bit depth, clipped pixels and the scale, so you know whether the calibration survived the export.
4. Measure in the **Measure** tab; lengths and areas come out in µm or nm with no Set Scale step.

:::grid
![Length and area in calibrated units in LabCanvas Image Analysis](https://www.labcanvas.com/analysis-examples/measure.webp "Length and area in calibrated units: Three nanorods measured with the line tool, 418.3, 652.8 and 454.1 nm long, and a fourth outlined with the wand: area 14,356.3 nm², perimeter 975.8 nm 2.5 nm per pixel, from the file.")
![Image check in LabCanvas Image Analysis](https://www.labcanvas.com/analysis-examples/check.webp "Image check: All fine: nothing clipped brightest value 3,688, even illumination the background varies by 1.06×, sharpest at slice 8 of 15.")
![Z-stack projection in LabCanvas Image Analysis](https://www.labcanvas.com/analysis-examples/stack.webp "Z-stack projection: Maximum intensity projection of slices 1–15 of the bead Z-stack: the beads from every depth sharp in one 256 × 256 image.")
:::

Why it is a good choice for the exported file:

- **Nothing to install**, on any computer or tablet, and TIFF, BigTIFF and OME-TIFF (8-, 16- and 32-bit, stacks and hyperstacks) open with their calibration, as do ImageJ ROI files (.roi, RoiSet.zip).
- **An image check on opening** that flags a lost scale, clipping and JPEG compression before you measure.
- **ImageJ's names and measurements**, with a methods sentence under every table and **Send to Graphs** for the statistics.
- **Free with a LabCanvas account**; downloading files is part of LabCanvas Pro.

For the vendor files themselves, Fiji with Bio-Formats remains the tool: it reads them directly, handles multi-series projects and pyramids, and opens files larger than memory as virtual stacks. [Open Image Analysis](https://www.labcanvas.com/analysis.html), or read the [complete guide](https://www.labcanvas.com/blog/image-analysis-complete-guide) for every format it reads.

ImageJ and Fiji are independent open-source projects; LabCanvas is not affiliated with them.

## References

- Linkert M, Rueden CT, Allan C, Burel JM, Moore W, Patterson A, et al. (2010). Metadata matters: access to image data in the real world. *Journal of Cell Biology* 189, 777–782. [doi:10.1083/jcb.201004104](https://doi.org/10.1083/jcb.201004104)
- Schindelin J, Arganda-Carreras I, Frise E, et al. (2012). Fiji: an open-source platform for biological-image analysis. *Nature Methods* 9, 676–682. [doi:10.1038/nmeth.2019](https://doi.org/10.1038/nmeth.2019)

## Frequently asked questions

### How do I open a .czi file in ImageJ?

Use Fiji, which includes Bio-Formats: drag the .czi onto the Fiji window or use File › Import › Bio-Formats, choose View stack with: Hyperstack, and click OK. In plain ImageJ 1.x, first put bioformats_package.jar from the Bio-Formats download page in the plugins folder and restart; then use Plugins › Bio-Formats › Bio-Formats Importer.

### How do I open .nd2 files in ImageJ?

The same way as .czi: Bio-Formats reads Nikon NIS-Elements .nd2 files. In Fiji, File › Import › Bio-Formats (or File › Open); multipoint or multi-well acquisitions may appear as several series, so tick Open all series or pick the ones you need in the series list.

### Why does ImageJ say "Unsupported format or file not found"?

Plain ImageJ 1.x has no reader for that file type, usually because Bio-Formats is not installed, or the path is wrong. Install Bio-Formats (or use Fiji, which includes it), or export the image as TIFF or OME-TIFF from the acquisition software. If Bio-Formats is installed and the file still fails, update it: newer versions of a format often need a newer reader.

### Is Bio-Formats included in ImageJ?

It is included in Fiji, and kept up to date by the Fiji Updater. Plain ImageJ 1.x does not include it; you add it by dropping bioformats_package.jar into the plugins folder.

### Does Bio-Formats keep the scale (pixel size) of my image?

Yes, when the file stores it: the pixel size, slice spacing, frame interval and channel names are read from the metadata and set on the image. Check them in Image › Properties. Saving the result as TIFF or OME-TIFF keeps them; saving as JPEG or PNG does not.

### Can LabCanvas open .czi or .nd2 files?

No. LabCanvas Image Analysis reads TIFF, BigTIFF and OME-TIFF (with their pixel size, channels, slices and frames), PNG, JPEG, WebP, GIF and BMP. Export the file as OME-TIFF from the acquisition software or from Fiji, and it opens with its scale.

## Related links

- [How to set the scale in ImageJ](https://www.labcanvas.com/blog/imagej-scale-bar-set-scale): When the file has no pixel size
- [Fiji vs ImageJ](https://www.labcanvas.com/blog/fiji-vs-imagej): What Fiji adds, Bio-Formats included
- [Merge and split channels in ImageJ](https://www.labcanvas.com/blog/merge-channels-imagej): Once the channels are open
- [Maximum intensity projection](https://www.labcanvas.com/blog/maximum-intensity-projection): Z-stacks from your confocal file
- [Microscope pixel size calculator](https://www.labcanvas.com/tools/microscope-pixel-size-calculator): Pixel size from camera, objective and binning
- [Image Analysis: the complete guide](https://www.labcanvas.com/blog/image-analysis-complete-guide): Which files open, and every tool